BIOCHEMICAL AND CELLULAR STUDIES OF QUIESCENT STIMULATED CULTURED CELLS

Abstract

Previous work from this laboratory has shown an antiproliferative effect of non-steroidal antiestrogens on estrogen receptor-negative mouse (EL4) and human (Raji) lymphoid cells. This thesis describes an antiproliferative effect in two other lymphoid cell lines (K36 and Ag-8) as well as in a lower eukaryote, the buckling yeast Saccharomyces cerevisiae, and in bacteria. The antiproliferative effect of antiestrogens in K36 cells was enhanced in lipoprotein-poor medium. The enhancement was not due to increased bioavailability of these compounds because cellular uptake of [3H]tamoxifen was not increased in lipoprotein-poor medium and because the lipoprotein fraction of serum had negligible [3H]tamoxifen-binding capacity. Cholesterol and lipoproteins, but not mevalonate, reversed the cytostatic effect of antiestrogens. Reversal by cholesterol was dose-related (10-7 M to 10-5 M), while that by lipoproteins could also be demonstrated in medium undepleted of lipoproteins. K36 cells were rich in intracellular antiestrogen binding sites (AEBS) (2946 ± 203 fn10l/mg protein). The cytostatic efficacy of a series of nonsteroidal antiestrogens and structurally related compounds correlated well with their relative binding affinities for solubilized antiestrogen-binding sites from K36 cells when log IC50 values (concentration required to reduce [3H]thymidine incorporation by 50%) were plotted against log RBA50 values (concentration required to reduce [3H]tamoxifen binding by 50%) (correlation coefficient 0.94). Transmission electron microscopy of antiestrogen-treated cells showed evidence of disordered cytokinesis which was partially reversed by cholesterol, and disruption of the Golgi appararus. From tumors induced by subcutaneous inoculation of K36 cells in syngenic AKR mice treated with clomiphene, variants of K36 with partial resistance to antiestrogens were isolated and characterized. One of these, CR-2, was morphologically similar to K36 but had generally enhanced resistance to antiestrogens. These resistant cells had a lower cellular AEBS content and accumulated 3-fold less [3H]tamoxifen (normalised for protein content) than K36 cells. The findings described in this thesis implicate the antiestrogen-binding protein in the antiproliferative effect of antiestrogens in nonestrogen target cells, and suggest links between nonsteroidal antiestrogen action and cholesterol or cholesterol metabolism.